1. Field of the Invention
The invention is directed toward culture plates used in the biomedical science field and, more specifically, to a culture plate having a membrane which may be flexed to stretch or compress cells in a monolayer or a three-dimensional culture adhering or tethered to the membrane.
2. Description of the Prior Art
In the human body, most cells are constantly subjected to tension and compression. Application of strain to cells in culture simulates the in vivo environment, causing dramatic morphologic changes and biomechanical responses in the cells. There are both long and short term changes that occur when cells are mechanically loaded in culture, such as alterations in the rate and amount of DNA or RNA synthesis or degradation, protein expression and secretion, the rate of cell division and alignment, changes in energy metabolism, changes in rates of macromolecular synthesis or degradation, and other changes in biochemistry and bioenergetics.
Methods of altering the mechanical environment or response of cells in culture have included wounding cells by scraping a monolayer, applying magnetic or electric fields, or by applying static or cyclic tension or compression with a screw device, hydraulic pressure, or weights directly to the cultured cells. Shear stress has also been induced by subjecting the cells to fluid flow. However, few of these procedures have allowed for quantitation of the applied strains or provided regulation to achieve a broad reproducible range of cyclic deformations.
A known device applies a defined, controlled static or variable duration cyclic tension or compression to growing cells in vitro. The device uses positive or negative pressure to deform a flexible membrane in the well of a culture plate thereby "exercising" the cells in culture and yielding up to 30% strain in the membranes. Vacuum is the preferred pressure modality. The device produces regulated strain to statically or cyclically stretch the rubber membrane and attached cells. This device is described in U.S. Pat. No. 4,789,601 issued Dec. 6, 1988 to the Applicant and is hereby incorporated by reference. With this device, the surface of the flexible membrane of the culture plate is treated to enable cells to adhere and grow thereupon. The culture plate membrane surface may include one of many materials including but not limited to genetic type I, II, III, IV collagens, elastin, fibronectin, laminen, peptides therefrom, integrin-binding peptides, compounds with amino or carboxyl functionalities, and/or combinations of peptides and proteins.
This flexing device allows users to simulate the mechanical load environment of walking, running, breathing or the beat of a heart, to cells cultured from mechanically active tissues, such as heart, lung, skeletal muscle, bone, ligament, tendon, cartilage, smooth muscle cells, endothelial cells and cells from other tissues. Rather than test the biological or biochemical responses of a cell in a static environment, the investigator can apply a frequency, amplitude and duration of tension or compression to cultured cells.
The membranes for the floors of the wells of culture dishes just described are made of blended silicone rubber. These rubber membranes are relatively thick (about three millimeters) and adhere to the side wall of the plastic culture dish by dry tack alone and are vertically supported in the culture dish by a lip extending radially from the side wall of the culture dish well. While the culture dish membrane can withstand millions of downward cyclic loadings, since it is supported by the lip and adheres to the sidewall, it may be loosened by an upward force. This is advantageous for easy removal and processing of the adherent cells. However, with this arrangement, upward cyclic loading is not permitted and the membrane can inadvertently become dislodged if such a loading is introduced.
Furthermore, while this device is satisfactory for a number of different applications, the thickness of the membrane only allows a gradient of strain upon distension by vacuum. The resulting nonuniform expansion of the membrane during use renders it less desirable than a more homogeneous strain field throughout the membrane. Additionally, this device is capable of accepting only negative pressure produced by a vacuum to deform the membrane downward but would not be amenable to positive pressure since this would tend to expel the membrane from the well. Moreover, the membrane is formed by pouring material into the wells in a culture plate and permitting it to cure. As a result, the physical shape and characteristic of each membrane could be slightly different depending upon the conditions of the individual silicone rubber lots, mixtures, pours, distribution along the culture plate sidewall, curing time and oven temperature.
In order to better control the expansion characteristics of the membrane caused by positive or negative pressures, it was desired to utilize a thin silicone membrane with or without a self-contained O-ring that fits into an O-ring cavity in the encompassing base and body of the plastic parts of the cell culture plate. The O-ring capture of the membrane permits precise pretensioning of the membrane within the well. Pretensioning prevents (1) the membrane from sagging when loaded with fluid culture medium, thus preventing cells from pooling in the well center during cell seeding, and (2) discontinuities in tension from one well to another, which would result in varying membrane strain when actively flexed. When the membrane of the prior art device was adhered to the well wall only by dry tack, such pretensioning was impossible.
An apparatus and method addressing these concerns is needed to yield reproducible unconstrained as well as constrained distension to the flexible membranes within the culture plate wells.